Cell migration is promoted by the alternating formation and disassembly of focal adhesions (FAs); our long-term goal is to understand the mechanisms that govern these dynamic events. In FA formation, multiple adhesion molecules are concentrated in a small area in which complex protein interactions are highly specific, although some of the binding affinities are relatively weak. Because of the relatively high protein concentration at the FA locus, binding affinity plays a critical role, and phosphorylation-mediated changes in binding affinity can completely alter the balance of interactions within the complex network. Therefore, to elucidate the mechanism that regulates the formation and disassembly of focal adhesions, we must understand in detail the structural and biophysical interactions within FAs and their binding partners and determine how phosphorylation affects these interactions; this is the focus of the project. Specifically, we will study paxillin family proteins and their biding partners in FAs. Paxillin and its related proteins are key players in FAs; as scaffold proteins, they use their N-terminal LD motifs to interact with other FA molecules. One major group of LD binding partners comprises the focal adhesion targeting (FAT) domain of FAK and the FAT-like domains of other FA proteins. Although the FAT-like domains have similar structures, their interactions with the LD motifs of paxillin family proteins differ in many ways. These differences offer an opportune starting point to dissect the dynamics of the complex interactions in FA formation and disassembly. This study will also aid in the design of inhibitors that target specifi binding events in FAs in order to mediate FA dynamics. PUBLIC HEALTH RELEVANCE: Cell migration is promoted by the alternating formation and disassembly of focal adhesions; our long-term goal is to understand the mechanisms that govern these dynamic events. Inappropriate regulations of focal adhesion dynamics have been implicated in cancer and other human diseases. Therefore, the knowledge obtained from the studies in the current project will not only help to explain the fundamental chemical nature of the complex molecular interplay in focal adhesions but may also lead to the development of therapeutic agents that target pathogenic abnormalities in FA signaling.